Human navigation relies on numerous cognitive operations, including location representation and route sequence memory. Using a memory of the spatial layout of locations and landmarks to guide behavior might be expected to draw heavily on spatial working memory (WM), whereas it has been suggested that route-based navigation draws upon a sequential memory representation. In previous work we have shown individual differences in the tendency to use these two navigational strategies. In the present study, we examined the contribution of working memory for spatial layout or a sequence on navigation, using a concurrent secondary task as participants navigated though a virtual environment. Participants (n = 60) learned to navigate from a start location to a target using a fixed route, and were then placed at a different location and asked to navigate to the previously learned target. During these probe recall trials, participants were randomly assigned to concurrently perform an auditory spatial WM task, an auditory sequence WM task, or a control task. We divided participants into those utilizing either a sequence-based or spatial-based strategy according to target marking with respect to the trained route or spatial layout of the environment. Spatial-based participants were differentially impacted by both secondary tasks; concurrent sequence WM led participants to consistently misplace the target location by a small but significant margin, whereas concurrent spatial WM significantly increased between-subject variability of target location. Notably, sequence-based participants were not disrupted by either secondary task. These results suggest that sequence-based memory is easier to maintain during any secondary task, while spatial-based memory is sensitive to interference from both spatial and sequence WM tasks. However, based on the degree of target mislocation, the attentional requirement of a concurrent spatial WM task has a greater impact on spatial-based navigational strategies than a sequential WM task.